This project is focused on the elucidation of molecular signal transduction mechanisms involved in activation and/or modification of NF-kappaB activity and on the mechanisms by which NF-kappaB regulates important target genes. This project complements other projects in which we aim to identify and characterize novel in vivo functions of NF-kappaB and its regulators. The NF-kappaB family of transcription factors is central to the regulation of both adaptive and innate immune responses. Once activated by stress- and pathogen-derived signals, these factors are responsible for executing genetic programs required in defense of the host against pathogenic insults. In addition, NF-kappaB factors have many other roles to maintain health; for example, they contribute to the development of the immune system. In contrast to essential tasks in health, aberrant NF-kappaB activity often plays critical roles in initiating and/or driving disease processes. The project is intended not only to lead to a better basic understanding of signaling processes that activate NF-kappB and the mechanisms by which it regulates genes in turn, but, in addition, to provide a basis for new approaches to treating diseases. Diseases in which NF-kappaB makes critical contributions include inflammatory and autoimmune diseases, as well as many types of cancer. For example, NF-kappaB can drive inflammatory processes that indirectly support tumor development of nearby cells, such as epithelial cells, or it may directly support tumorigenesis by ensuring the survival of the afflicted cell, or both. Consequently, NF-kappaB and the network of signaling proteins that control the activity of these transcription factors become potential targets for therapeutic interventions. We have previously identified an adaptor protein named CIKS, which associates with the IkappaB kinase (IKK) complex; the IKK complex activates NF-kappaB in response to many inflammatory signals, and this is known as the classical pathway of activation. In FY 2008 we have shown that the IL-17 cytokine transmits all of its signals via the adaptor protein CIKS, including the activation of NF-kappaB. We have further shown that the activation of NF-kappaB is essential for IL-17 induced expression of select, but not all target genes. In FY 2008 we have generated mutants of CIKS, which will be assessed for their potential to transmit IL-17 signals. The initial aim is to pinpoint short seqments of CIKS that are critical for signaling. Ultimately we anticipate making use of this information to generate possible inhibitors of this pathway. In addition to the classical pathway, NF-kappaB can also be activated via an alternative or non-classical pathway, independent of IKK; this involves processing of the NF-kappaB2 p100 precursor to p52 to allow for nuclear entry of the p52/RelB NF-kappaB heterodimers. We have previously determined that the TNF family member BAFF provides a survival signal to B cells via activation of the alternative pathway. In addition, we have shown that Lymphotoxin beta, CD40 ligand and RANK ligand activate the alternative pathway to help differentiate stromal cells and to empower these cells to organize lymphoid organs. We are investigating the roles of the TRAF3 and TRAF2 adaptors in this pathway. In FY 2008 we have generated a series of TRAF2 and TRAF3 mutants, which will be used to decipher the functions of subdomains of these proteins. Bcl-3 is an IkappaB-like protein that can positively and negatively regulate gene activity via interaction with specific NF-kappaB complexes in the nucleus. In FY 2008 we have determined that loss of Bcl-3 in macrophages and dendritic cells changes their cytokine expression profile in response to the endotoxin LPS. We are investigating the underlying mechanisms.